pone.0134896.g005: LMP1 causes a high glycolytic level and induces vulnerability to DPI.A: Lactate production in NP69 and NP69-LMP1 cells. Cells were incubated in KSF medium at a density of 5 × 105 cells/mL for 24 hr. The lactate concentration in the medium was measured using an Accutrend Lactate Analyzer as described in Materials and Methods (mean ± SD of three experiments, * p<0.05). B: Comparison of glucose uptake in NP69 and NP69-LMP1 cells for 1 hr. Cells were incubated in glucose-free RPMI 1640 for 2 hr. Glucose uptake was detected by flow cytometry using a fluorescent deoxyglucose analog (2-NBDG). Each histogram is representative of three experiments. C: Oxygen consumption in NP69 and NP69-LMP1 cells (mean ± SD of three experiments, p = 0.5106). D: Comparison of lactate generation (left panel) and glucose consumption (right panel) in NP69-pZIPNeoSV(X)1 and NP69-pZIPNeoSV(X)1-LMP1 transient-transfected cells. Medium lactate and glucose concentration (nmol/L per 105 cells, mean ± SD of three experiments, * p<0.05) were measured as described in Materials and Methods. E: An inhibitor of glucose uptake, 3-bromopyruvate, was used to test the glucose uptake in NP69 and NP69-LMP1 cells. Each histogram is representative of three experiments. F: Effect of combining DPI and 3-bromopyruvate on lactate production in NP69-LMP1 cells. A volume of 30 μM 3-bromopyruvate completely inhibited lactate production in NP69-LMP1 cells. When combined with 3-bromopyruvate, 5 μM DPI treatment could not induce any increase in lactate (mean ± SD of three experiments). * p<0.05, DPI treated group vs untreated control group; ** p<0.05, 3-BrOP treated group alone or combined with DPI vs DPI treated group. G: Treatment with 0.3–30 μM DPI preferentially killed NP69-LMP1 cells compared to NP69 cells (mean ± SD of three experiments). H: Preferential killing of NP69-LMP1 cells by 5 μM DPI was detected by annexin V-PI staining and flow cytometry. The numbers under the plots indicate live cells with both low annexin V staining and low PI staining. Each histogram is representative of three experiments.

Mentions:
Under hypoxia and oxidative stress, cancer cells are more dependent on anaerobic respiration and the glycolytic pathway to meet excessive bioenergetics needs [17]. To evaluate the influence of LMP1-induced oxidative stress on energy metabolism, we examined the glycolytic activity in NP69 and NP69-LMP1 cells. Glycolytic activity can be evaluated using the glycolytic index as calculated using the following formula: (lactate generation rate × glucose uptake rate) / oxygen consumption rate [9]. As shown in Fig 5A and 5B, compared with NP69 cells, NP69-LMP1 cells produced more lactate (1.4-fold increase, p = 0.02) and exhibited a increased basal level of glucose uptake (1.5-fold) as quantified by flow cytometry using 2-NBDG. However, compared with NP69 cells, NP69-LMP1 cells did not exhibit an obvious decrease in oxygen consumption (Fig 5C) Increased glucose uptake and lactate accumulation indicate increased glycolytic activity in NP69-LMP1 cells, with a greater than 2-fold increased glycolytic index compared with NP69 cells. Additionally, using a transient transfection system (Fig 5D), we found that, pZIPNeoSV(X)1-LMP1 transfection significantly increased both lactate generation (approximately 4.57-folds increase) and glucose consumption as demonstrated by an approximately 65% reduction in glucose the medium compared with NP69 cells transfected with empty vector (p < 0.001). The PI3K/Akt pathway directs cancer cells towards aerobic glycolysis by activating the c-Myc and mTOR pathways [18,19]. Furthermore, LMP1 activates PI3K/Akt signaling in LMP1-mediated transformation [20], which suggests that LMP1 might activate glycolysis via the Akt pathway.

pone.0134896.g005: LMP1 causes a high glycolytic level and induces vulnerability to DPI.A: Lactate production in NP69 and NP69-LMP1 cells. Cells were incubated in KSF medium at a density of 5 × 105 cells/mL for 24 hr. The lactate concentration in the medium was measured using an Accutrend Lactate Analyzer as described in Materials and Methods (mean ± SD of three experiments, * p<0.05). B: Comparison of glucose uptake in NP69 and NP69-LMP1 cells for 1 hr. Cells were incubated in glucose-free RPMI 1640 for 2 hr. Glucose uptake was detected by flow cytometry using a fluorescent deoxyglucose analog (2-NBDG). Each histogram is representative of three experiments. C: Oxygen consumption in NP69 and NP69-LMP1 cells (mean ± SD of three experiments, p = 0.5106). D: Comparison of lactate generation (left panel) and glucose consumption (right panel) in NP69-pZIPNeoSV(X)1 and NP69-pZIPNeoSV(X)1-LMP1 transient-transfected cells. Medium lactate and glucose concentration (nmol/L per 105 cells, mean ± SD of three experiments, * p<0.05) were measured as described in Materials and Methods. E: An inhibitor of glucose uptake, 3-bromopyruvate, was used to test the glucose uptake in NP69 and NP69-LMP1 cells. Each histogram is representative of three experiments. F: Effect of combining DPI and 3-bromopyruvate on lactate production in NP69-LMP1 cells. A volume of 30 μM 3-bromopyruvate completely inhibited lactate production in NP69-LMP1 cells. When combined with 3-bromopyruvate, 5 μM DPI treatment could not induce any increase in lactate (mean ± SD of three experiments). * p<0.05, DPI treated group vs untreated control group; ** p<0.05, 3-BrOP treated group alone or combined with DPI vs DPI treated group. G: Treatment with 0.3–30 μM DPI preferentially killed NP69-LMP1 cells compared to NP69 cells (mean ± SD of three experiments). H: Preferential killing of NP69-LMP1 cells by 5 μM DPI was detected by annexin V-PI staining and flow cytometry. The numbers under the plots indicate live cells with both low annexin V staining and low PI staining. Each histogram is representative of three experiments.

Mentions:
Under hypoxia and oxidative stress, cancer cells are more dependent on anaerobic respiration and the glycolytic pathway to meet excessive bioenergetics needs [17]. To evaluate the influence of LMP1-induced oxidative stress on energy metabolism, we examined the glycolytic activity in NP69 and NP69-LMP1 cells. Glycolytic activity can be evaluated using the glycolytic index as calculated using the following formula: (lactate generation rate × glucose uptake rate) / oxygen consumption rate [9]. As shown in Fig 5A and 5B, compared with NP69 cells, NP69-LMP1 cells produced more lactate (1.4-fold increase, p = 0.02) and exhibited a increased basal level of glucose uptake (1.5-fold) as quantified by flow cytometry using 2-NBDG. However, compared with NP69 cells, NP69-LMP1 cells did not exhibit an obvious decrease in oxygen consumption (Fig 5C) Increased glucose uptake and lactate accumulation indicate increased glycolytic activity in NP69-LMP1 cells, with a greater than 2-fold increased glycolytic index compared with NP69 cells. Additionally, using a transient transfection system (Fig 5D), we found that, pZIPNeoSV(X)1-LMP1 transfection significantly increased both lactate generation (approximately 4.57-folds increase) and glucose consumption as demonstrated by an approximately 65% reduction in glucose the medium compared with NP69 cells transfected with empty vector (p < 0.001). The PI3K/Akt pathway directs cancer cells towards aerobic glycolysis by activating the c-Myc and mTOR pathways [18,19]. Furthermore, LMP1 activates PI3K/Akt signaling in LMP1-mediated transformation [20], which suggests that LMP1 might activate glycolysis via the Akt pathway.

Bottom Line:
In this study, we used LMP1-transformed NP cells and EBV-related malignant cell lines to assess the effects of LMP1 on reactive oxygen species (ROS) accumulation and glycolytic activity.Additionally, in both NPC cells and tissue samples, p22phox expression correlated with LMP1 expression.The NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI) also exerted a marked cytotoxic effect in LMP1-transformed and malignant cells, providing a novel strategy for anticancer therapy.